Gram-negative bacterial infections remain an enormous public health challenge. Failure to control infection can lead to sepsis, a severe pathology driven by dysregulated immune responses to lipopolysaccharide (LPS) and other microbial products. Sepsis can progress to multi-organ failure, a severe drop in blood pressure, and septic shock. Gram-negative infections are responsible for over 10 million cases of sepsis worldwide each year, with a greater than 30% mortality rate. Critically, more than a hundred clinical trials of immunomodulators that successfully treat sepsis in mice have failed, resulting i a shortage of effective treatments for human sepsis. The basis for these failures is unclear, but fundamental differences in human and mouse innate immune responses to infection likely play an important role. Our long-term goal is to elucidate the molecular mechanisms underlying human-specific innate immune responses to infection, as this knowledge is essential for developing new treatments for sepsis. To this end, we study the gram-negative pathogen Legionella pneumophila. Legionella causes the severe pneumonia Legionnaires' disease, which can develop into sepsis if not promptly treated. To initiate disease, Legionella infects and replicates within macrophages by delivering bacterial virulence factors into the host cell cytosol. Cytosolic immune detection of translocated bacterial products triggers assembly of inflammasomes, multiprotein complexes that activate caspases to induce host cell death and release of IL-1 family cytokines. We and other groups recently identified two types of inflammasomes in murine cells that respond to Legionella and other gram-negative pathogens: canonical inflammasomes activate caspase-1 (CASP1), while noncanonical inflammasomes engage caspase-11 (mCASP11), which directly detects cytosolic LPS. Although mCASP11 is critical for host defense, mCASP11 also mediates endotoxic shock. Intriguingly, humans express two mCASP11 orthologs, hCASP4 and hCASP5, both of which also recognize LPS, but how they control inflammasome responses to bacterial infection is poorly understood. Our recently published and new findings reveal major differences in mouse and human noncanonical inflammasomes and indicate that hCASP4 and hCASP5 have distinct roles. We thus hypothesize that hCASP4- and hCASP5-mediated responses to bacterial infection drive human-specific inflammatory responses. Thus, we will pursue two aims that will examine how hCASP4 and hCASP5 regulate human inflammasome responses to infection and define the bacterial and host factors required for noncanonical inflammasome activation in primary human macrophages. These studies will provide fundamental insight into how noncanonical inflammasomes function in human cells, and will shed critical light on human-specific mechanisms that regulate anti-bacterial immune responses and sepsis.